Abstract
Galleria mellonella larvae are an alternative in vivo model that has been extensively used to study the virulence and pathogenicity of different bacteria due to its practicality and lack of ethical constraints. However, the larvae possess intrinsic autofluorescence that obstructs the use of fluorescent proteins to study bacterial infections, hence better methodologies are needed. Here, we report the construction of a promoter probe vector with bioluminescence expression as well as the optimization of a total bacterial RNA extraction protocol to enhance the monitoring of in vivo infections. By employing the vector to construct different gene promoter fusions, variable gene expression levels were efficiently measured in G. mellonella larvae at various time points during the course of infection and without much manipulation of the larvae. Additionally, our optimized RNA extraction protocol facilitates the study of transcriptional gene levels during an in vivo infection. The proposed methodologies will greatly benefit bacterial infection studies as they can contribute to a better understanding of the in vivo infection processes and pathogen–mammalian host interactions.
Highlights
Galleria mellonella is a popular animal model for the study of virulence and pathogenicity of different bacteria
G. mellonella larvae were injected with an infective dose of P. aeruginosa PAO1 wild-type and P. aeruginosa PAO1 containing plasmids derived from pETS130 (Table S1), which encode transcriptional fusions of the nrdA and nrdJ promoters with green fluorescent protein (GFP) and E2Crimson that were previously constructed in our laboratory [14,19]
G. mellonella larvae were injected with an infective dose of P. aeruginosa PAO1 wild-type, P. aeruginosa PAO1 expressing GFP (PAO1::eGFP), and P. aeruginosa PAO1 expressing the luxCDABE genes (PAO1::lux) followed by incubation at 37 ◦C for about 19 h or until death
Summary
Galleria mellonella (greater wax moth) is a popular animal model for the study of virulence and pathogenicity of different bacteria. With an innate immune response very similar to the one found in mammals, G. mellonella larvae possess both cellular and humoral defenses [3] They are widely used for testing the efficacy and toxicity of new antimicrobial agents, offering an additional pre-screening model to lower the number of drug tests performed in mammalian animals [4,5]. The use of green and red fluorophores is associated with background autofluorescence problems that hinder the real measurement of bacterial fluorescence For this reason, bioluminescence currently seems like the best methodology alternative to monitor an in vivo infection in G. mellonella. We report the construction and utilization of several lux vectors containing different nrd promoter fusions as a better approach to study and monitor a P. aeruginosa in vivo infection in Galleria mellonella. The optimized methodologies will be valuable for the study of different infections and further applications in G. mellonella
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